U.S. patent number 4,090,022 [Application Number 05/779,950] was granted by the patent office on 1978-05-16 for porous cellulose beads.
This patent grant is currently assigned to Purdue Research Foundation. Invention is credited to Li Fu Chen, George T. Tsao.
United States Patent |
4,090,022 |
Tsao , et al. |
* May 16, 1978 |
Porous cellulose beads
Abstract
Porous cellulose beads are prepared by distributing droplets of
a solvent mixture containing a cellulose derivative into a
precipitating solution to form porous beads which are then washed
and hydrolyzed to form porous cellulose beads. The porous cellulose
beads, which may be cross-linked, if desired, by suitable
treatment, are useful carriers to which enzymes can be immobilized.
The beads may also be used for the separation of enzymes, proteins,
nucleic acids and the like, or to remove metal ions from dilute
mining solutions.
Inventors: |
Tsao; George T. (West
Lafayette, IN), Chen; Li Fu (West Lafayette, IN) |
Assignee: |
Purdue Research Foundation
(West Lafayette, IN)
|
[*] Notice: |
The portion of the term of this patent
subsequent to December 13, 1994 has been disclaimed. |
Family
ID: |
24727136 |
Appl.
No.: |
05/779,950 |
Filed: |
March 21, 1977 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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679497 |
Apr 22, 1976 |
|
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Current U.S.
Class: |
536/57; 264/13;
264/15; 264/14; 435/179; 530/413; 530/416; 530/814; 536/80; 536/98;
536/99 |
Current CPC
Class: |
C08J
9/28 (20130101); C12N 11/12 (20130101); C08J
2201/0542 (20130101); Y10S 530/814 (20130101); C08J
2301/00 (20130101) |
Current International
Class: |
C12N
11/00 (20060101); C12N 11/12 (20060101); C08J
9/28 (20060101); C08J 9/00 (20060101); C08B
015/10 (); C08B 016/00 () |
Field of
Search: |
;195/63,DIG.11
;264/13-15 ;536/57,80,98,99 ;260/13 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Tsumura et al., "Continuous Isomerization of Glucose by a Column of
Glucose Isomerase," Journal of Food Science and Technology, vol.
14, No. 12, pp. 539-540 (1967)..
|
Primary Examiner: Griffin; Ronald W.
Attorney, Agent or Firm: Cushman, Darby & Cushman
Parent Case Text
This application is a continuation-in-part of our co-pending
application Ser. No. 679,497 filed Apr. 22, 1976.
Claims
We claim:
1. A process for the preparation of porous cellulose beads suitable
for use as a carrier of enzymes and other biological agents which
comprises the steps of:
a. dissolving a hydrolyzable cellulose derivative in an inert
organic, water-miscible solvent to form a solution having a density
greater than that of a precipitation solution the cellulose
derivative to solvent ratio ranging from 1:20 to 1:3
weight/volume;
b. distributing said solution in the form of droplets into a
precipitation solution whereby said cellulose derivative is
precipitated in the form of uniformly porous beads;
c. separating the precipitated beads from said solution;
d. washing the separated porous beads with water;
e. hydrolyzing the washed beads to convert the beads to cellulose
and to increase the active sites for attachment of enzymes and
other biological agents;
f. washing the hydrolyzed beads to obtain porous cellulose beads
having a uniformly distributed void space greater than 50% by
volume.
2. A process according to claim 1 wherein distributing is
accomplished by spraying.
3. A process according to claim 1 wherein said precipitation
solution is selected from the group consisting of water, hexane,
cyclohexane, octane, benzene and mixtures of water and ethanol or
methanol.
4. A process according to claim 3 wherein said precipitation
solution is water.
5. A process according to claim 1 wherein said cellulose derivative
is cellulose acetate and hydrolysis is carried out in a caustic
solution.
6. A process according to claim 1 wherein said solvent is a mixture
of:
a. a member from the group consisting of acetone, a mixture of
acetone and methanol or ethanol, methyl acetate, a mixture of
methylene dichloride and methanol, methyl ethyl ketone, formamide
and dimethyl sulfoxide; and
b. a member from the group consisting of dimethyl sulfoxide,
formamide, methyl acetate, cyclohexanone, methylene dichloride,
ethylene dichloride, a mixture of methylene dichloride and
methanol, and a mixture of ethylene dichloride and methanol.
7. A process according to claim 6 wherein said solvent is dimethyl
sulfoxide, formamide or methyl acetate.
8. A process according to claim 1 wherein the void space of said
beads is from about 75 to 95%.
9. A process according to claim 1 wherein said porous cellulose
beads are cross-linked with at least one cross-linking agent to
obtain cross-linked porous cellulose beads.
10. A process according to claim 9 wherein said beads are
cross-linked prior to being hydrolyzed.
11. A process according to claim 9 wherein said beads are
cross-linked after being hydrolyzed.
12. A process according to claim 9 wherein said cross-linking agent
is a diisocyanate.
13. A process according to claim 12 wherein said diisocyanate is
tolylene-2,4-diisocyanate or hexamethylene diisocyanate.
14. A process according to claim 9 wherein said cross-linking agent
is epichlorohydrin in a sodium hydroxide solution.
15. A process according to claim 9 wherein said cross-linking agent
is formaldehyde in a hydrochloric acid solution.
16. A process according to claim 9 wherein said cross-linking agent
is glutaraldehyde.
17. Porous cellulose beads produced according to the process of
claim 1.
18. Porous cross-linked cellulose beads produced according to the
method of claim 9.
19. A method for the removal of metallic ions from a dilute
solution containing said ions which comprises contacting the dilute
solution with the porous cellulose beads of claim 17, said beads
being further characterized by the presence of functional groups
capable of attaching said ions to the beads.
20. A method for the separation and purification of enzymes,
proteins, and nucleic acids which comprises contacting enzymes,
proteins or nucleic acids with the porous beads of claim 17, said
beads being further characterized by the presence of functional
groups capable of attaching said enzymes, proteins or nucleic
acids.
Description
BACKGROUND OF THE INVENTION
Porous cellulose beads provide a relatively lowcost, stable
material possessing versatile chemical properties such that thay
can be useful as a carrier for immobilized enzymes and other active
biological agents.
While ordinary cellulose particles and regenerated cellulose
powders meet most of the desired requirements of good carriers to
which enzymes can be immobilized, they suffer from configural
disadvantages which cause column reactors to become tightly packed
resulting in reduction of flow and sometimes channeling, and thus
insufficient contact between the immobilized enzyme and reaction
fluid. The immobilization of enzymes on an insoluble carrier is a
widely-accepted technique for a practical application of enzymes,
avoiding the necessity of employing fresh enzymes for each desired
use. Through immobilization of the enzyme, stabilization is
achieved which provides for efficient enzyme use and provides for
the design and operation of enzyme reactors in a continuous
mode.
To a large degree, the success of an immoblized enzyme for use in
practical application depends upon the properties of the carriers
employed for immoblization. Accordingly, a good carrier should meet
the requirements of being inexpensive and should be of such a
physical shape that it is easy to be employed in reactors. In this
regard, the shape of a spherical bead is particularly desirable,
since it is useful in a packed bed, fluidized bed, expanded bed,
stirred tank, or other common types of chemical reactor designs.
Such a carrier should also have the proper physical and mechanical
strength such that it will not be crushed or deformed when packed
in a tall column. Crushing and deformation results in the column
becoming tightly packed, thereby blocking the flow of liquid
reagents through the column, thus decreasing the efficiency of the
chemical reactor. Suitable carriers should also possess versatile
chemical properties such that the immoblization of enzymes and
other biological agents onto the carrier through ionic or chemical
covalent bonding, as well as surface absorption, can be readily
achieved. In this regard, the carrier should have a high capacity
for forming a large number of bonds such that each unit of the
carrier can immobilize large amounts of the enzyme desired. Thus, a
carrier having a high degree of porosity and uniformly distributed
internal void spaces is particularly desirable. Such porosity
provides for good diffusion of chemical reagents or reaction
products into and out of the internal void spaces of the cellulose
beads. Carriers should be chemically stable, physically strong, and
made of inert material which resists microbiological attack causing
carrier deterioration in order to provide an immobilized enzyme
system having a prolonged active life.
Currently, porous glass and porous ceramic particles are commonly
employed for the immoblization of enzymes and while such particles
meet most of the above requirements for an acceptable particle,
they are relatively expensive. Furthermore, the number of chemical
reactions which may be used for immobilization of enzymes to glass
and ceramic carriers is limited.
In U.S. Pat. Nos. 3,947,325; 3,905,954; 3,573,277; 3,505,299;
3,501,419; 3,397,198; 3,296,000; 3,251,824; 3,236,669; 2,843,583;
2,773,027; 2,543,928 and 2,465,343, there is described the
preparation of a variety of cellulose materials in a variety of
forms, some of which are described as suitable for use in fixing
biologically-active materials such as enzymes or ion-exchange
groups thereto. However, these processes seem to suffer also from
the disadvantage of being expensive and the products obtained
generally are of an undesirable physical shape for use in such
chemical reactors as packed beds and fluidized beds. In particular,
the prior art fails to provide a means for producing spherical
shaped cellulose beads having a uniform distribution of pores
throughout the surface and a large uniformly porous internal void
space. Furthermore, the cellulose particles and powders of the
prior art generally are of such a small particle size that they are
not suited for use in chemical reactors. In addition, the cellulose
powders and particles of the prior art often have a hard surface
skin which causes severe diffusional hinderance and inefficient use
in chemical reactors.
In our earlier application, we describe the process of making
highly porous cellulose beads of uniform porosity which were found
highly suitable for immobilizing enzymes. We have found that these
beads may also be useful in the purification and separation of
enzymes, proteins, nucleic acids and the like. Furthermore, the
beads may be useful to separate metallic ions from dilute solutions
containing same.
Accordingly, the primary object of the present invention is to
provide a means for preparing inexpensive, highly-porous, stable
particles having versatile chemical properties whereby they may be
useful as a carrier to which enzymes or other biologically-active
materials can be immoblized.
A further object of the present invention is to provide a method
for the transformation of cellulose derivatives into highly-porous
particles having good mechanical stability such that it will
provide for adequate passage of liquid therethrough when operated
in packed bed reactors.
Still yet another object of the present invention is to provide a
porous cellulose bead having sufficiently large surface area to
provide high immobilization capacity of enzymes.
Still a further object of the present invention is to provide a
porous cellulose bead having improved physical and mechanical
strength so that it will not be crushed and deformed when used in
chemical reactors.
Yet a further object of the invention is to provide an improved
means for the purification and/or separation of enzymes, proteins,
nucleic acids and the like.
Yet another object of our invention is to provide a means for the
separation of metallic ions from dilute solutions containing
same.
These and other objects of the present invention will be more fully
apparent from the discussion set forth hereinbelow.
DESCRIPTION OF THE INVENTION
According to the present invention, a process is provided for the
preparation of porous cellulose beads which are suitable for use as
a carrier of enzymes and other biological agents. The invention
also provides a means for the modification of the chemical and
physical property of porous beads made from cellulose derivatives,
as well as techniques for immobilizing enzymes and other biological
active agents onto the porous beads so formed. While orginary
microcrystalline cellulose and other particles made from cellulose
satisfy many of the general requirements for a suitable carrier of
enzymes, such particles suffer from the tendency to pack together
tightly under pressure and also fail to provide sufficient porosity
to attach a sufficiently-large amount of enzymes thereto. Cellulose
derivatives are generally inexpensive and when treated according to
our invention provide a highly-versatile material for chemical
reactions being generally biologically inert. Thus, the cellulose
derivative beads herein provide many desirable properties for use
as a carrier of immobilized enzymes.
Our process for the modification of the physical properties of
cellulose derivatives, in order to produce porous cellulose beads,
involves the steps of:
a. dissolving a cellulose derivative in an inert organic,
water-miscible solvent to form a solution having a density greater
than that of the precipitation solution as defined hereinbelow;
b. distributing said solution in the form of droplets into a
precipitation solution whereby said cellulose derivative is
precipitated in the form of uniformly porous beads;
c. separating the precipitated beads from said solution;
d. washing the separated porous beads with water;
e. hydrolyzing the washed beads to convert the beads to cellulose
and to increase the active sites for attachment of enzymes and
other biological agents;
f. washing the hydrolyzed beads to obtain porous cellulose
beads.
According to the present invention, by dissolving a cellulose
derivative in a selected solvent and distributing same into a
selected precipitation solution, we are able to produce cellulose
beads of high uniform porosity and superior chemical and physical
properties. The beads produced in accordance with the present
invention are highly porous. The pores are generally uniformly
distributed over the surface and throughout the interior of the
bead. By proper selection of solvents and precipitation solutions,
the pore size of the beads may be controlled. It is of particular
advantage that in accordance with the process we are able to
control both the pore size and pore distribution. With reference to
FIGS. 2, 4(A) and (B), it will be seen that the pore openings are
uniformly distributed over the surface of the bead and were
estimated to be about 1,000 A which is a proper size for movement
of enzyme and reagent molecules in the pores.
The inert organic water miscible solvent may be a single liquid or
a combination of liquids. It is important that one employ a correct
combination of inert organic solvent and precipitation solution in
order to obtain the porous cellulose beads of desired shape and
pososity. The inert organic water-miscible solvent may be a
combination of liquids which together with the cellulose derivative
provide a solution which when mixed with the precipitating solution
results in a phase inversion whereby the cellulose derivative is
coagulated in the form of a porous bead. The inert organic solvent
thus contains a component (a) which is characterized as a liquid
which is capable of dissolving the cellulose derivative, such as
cellulose acetate, and is soluble in the precipitation
solution.
A second component (b) of the solvent system is a liquid which is
soluble in component (a) and also in the precipitation solution and
which is present in the solvent solution in an amount sufficient
that the density of the final solvent solution (together with the
cellulose derivative) is sufficiently higher than the density of
the precipitation solution so that upon distributing the solvent
solution in the form of droplets into the precipitation solution
the cellulose will coagulate and precipitate out as a porous bead
of desired size and porosity. Component (b) of the solvent is used
to control the surface activity of the solvent solution such that
the droplets of solvent solution will maintain their shape upon
contact with the precipitation solution. Component (b) also serves
to control the pore size and porosity of the precipitated beads. In
some instances, component (a) and component (b) may be the same. In
other instances, it may be appropriate to employ one or more
liquids in preparing component (a) and/or component (b).
As used herein, the term "precipitation solution" is defined as a
liquid solution which is a non-solvent for the cellulose derivative
and is miscible with the above inert organic, water-miscible
solvent. By means of illustration, the precipitation solution may
be water or an aqueous solution. The precipitation solution thus is
miscible with both solvent components (a) and (b). Thus, it will be
appreciated that when one dissolves the cellulose derivative in the
organic solvent, and subsequently adds a drop of the resulting
solvent solution to the precipitation solution, the cellulose
derivative will coagulate and precipitate out due to the phase
inversion which the cellulose derivative undergoes thereby forming
the desired porous cellulose bead.
As will be apparent from the discussion herein, a number of
variations are possible in the above-described process in preparing
the desired porous cellulose beads. In addition to cellulose
acetate, other cellulose derivatives may be employed as a starting
material for the preparation of the porous beads, for example,
cellulose nitrate and methyl cellulose. The terms "cellulose
derivative" and "hydrolyzable cellulose derivative" as used herein
are intended to include materials from which cellulose may be
regenerated such as by means of, for example, hydrolysis or
hydrogenation.
The organic solvent components (a) and (b) for the cellulose
derivative can vary, but should be chemically inert to the
cellulose derivative and wholly or substantially miscible with the
precipitation solution. It is of prime importance that the density
of the solvent solution formed by adding the cellulose derivative
to the inert solvent be greater than that of the precipitation
solution into which it is distributed such that when droplets of
the solvent solution are distributed into the precipitation
solution, the droplets will sink when the aqueous solution is not
agitated. Suitable single solvents, when using an aqueous
precipitation solution, include among others, for example,
dimethylsulfoxide and methyl acetate. It should be understood that
commercially available materials may be employed as solvent
components (a) and/or (b), and that these materials may contain
moisture, which in some instances has been found to be
advantageous.
When employing an aqueous precipitation solution, one may suitable
use as solvent component (a) a member from the group consisting of
acetone, formamide a mixture of acetone and methanol or ethanol,
methyl acetate, a mixture of methylene dichloride and methanol,
methyl ethyl ketone and dimethyl sulfoxide. The solvent component
(b) may thus be suitably chosen from a member selected from the
group consisting of dimethyl sulfoxide, formamide, methyl acetate,
cyclohexanone, methylene dichloride, ethylene dichloride, a mixture
of methylene dichloride and methanol, and a mixture of ethylene
dichloride and methanol.
A preferred solvent component (a) is acetone, but other solvents
can be suitably employed, and when using an aqueous precipitation
solution one may select a component (a) from the following
materials (the ratio of mixtures being the minimum ratio desirable
on a volume basis):
______________________________________ Component Minimum Ratio (a)
(Volume) ______________________________________ Acetone -- Acetone
+ Methanol 60:40 Acetone + Ethanol 60:40 Methyl acetate --
Methylene dichloride + Methanol 80:20 Dimethyl Sulfoxide -- Methyl
Ethyl Ketone -- Formamide --
______________________________________
As noted above, the primary function of component (a) is to
dissolve the cellulose derivative. The addition of component (b) is
necessary in order to provide a solvent solution having the
requisite density such that the cellulose derivative will
precipitate out in the precipitation solution. Component (b) also
provides for the control of pore size and uniform porosity of the
beads.
The solvent component (b) therefore provides for the desired
specific gravity of the solvent solution and when employing an
aqueous precipitation solution it is preferred to use dimethyl
sulfoxide as component (b). As will be appreciated, in some
instances component (a) and component (b) may be the same, i.e.
dimethyl sulfoxide, formamide or methyl acetate when used with
aqueous precipitation solutions. Various materials which may be
used suitably as component (b) when employing an aqueous
precipitation solution are outlined below.
______________________________________ Component Minimum Ratio (b)
(Volume) ______________________________________ Dimethyl sulfoxide
-- Ethylene dichloride + methanol 60:40 Methylene dichloride +
methanol 60:40 Ethylene dichloride -- Methylene dichloride --
Formamide -- Cyclohexanone --
______________________________________
The solution of cellulose derivative and inert solvent should have
a controlled cellulose derivative-to-solvent ratio since such will
have an effect on the eventual porosity of the beads prepared.
Generally, a small ratio (larger content of solvent) results in
beads having a larger porosity. A cellulose-to-solvent (including
components (a) and (b) ratio of from 1:20 and 1:3 (weight/volume)
has been found suitable for preparing cellulose beads having
various specific applications. Preferably, a cellulose
derivative-to-solvent ratio of 1:10 to 1:6 (weight/volume) is
employed to provide an easy-to-handle solution which results in
porous cellulose beads of desirable properties having a void space
of at least 50% by volume, preferably 75 to 95% and most suitably
about 75 to 80%. Beads having a higher porosity will generally have
a larger proportion of uniformly distributed internal void spaces
providing less diffusion hindrance, but will be somewhat weaker in
physical strength than beads of lower porosity.
The preferred precipitation solution into which the solution of
cellulose derivative is to be distributed generally consists of
water, but may be an aqueous solution which contains suitable
amounts of non-ionic or ionic surfactants to reduce the surface
tension thereof and facilitate formation of the porous beads. The
precipitation solution can also suitably contain a mixture of water
and methanol or ethanol (volume ratio 50:50). It is also envisioned
that the precipitation may be non-aqueous so long as the cellulose
derivative is insoluble therein and the necessary density
requirement is met. Thus, hydrocarbon solutions may be used such as
cyclohexane, hexane, decane, benzene and the like so long as they
are liquid in form, possess a density less than that of the inert
organic solvent and are miscible therewith. When the cellulose
derivative solution is distributed by spraying via a suitable means
such as a spray nozzle, the pressure drop and miscibility of the
inert solvent in the aqueous solution results in a dispersion and
ultimate precipitation of porous beads of the cellulose
derivative.
As will be appreciated by those skilled in the art, in
precipitating the cellulose beads, a sufficient amount of solvent
component (b) must be present in order that the solvent containing
cellulose derivative possess the requisite higher density than that
of the precipitation solution. Table 1 sets forth a number of inert
organic solvents for the precipitating of a cellulose derivative in
an aqueous solution. The ratios set forth are the minimum needed in
order to provide a solvent solution having a density greater than
that of water. As can be seen, the greater the specific gravity of
component (b), the less of that component is needed in order to
achieve the minimum density.
TABLE 1 ______________________________________ Minimum Solvent
Volume Ratio Component (a) Component (b) a:b
______________________________________ Acetone Dimethyl sulfoxide
70:30 Acetone Ethylene dichloride 80:20 Acetone Methylene
dichloride 80:20 Acetone Formamide 75:25 Acetone Cyclohexanone
45:55 Acetone Methyl acetate 35:65
______________________________________
After precipitation of the porous beads, cellulose is regenerated
from the derivative by hydrolysis in order to create more active
sites for enzyme attachment. In regenerating cellulose from its
derivative after formation of the beads, one can remove the
substituting groups (such as acetate from cellulose acetate) in
order to regenerate all the hydroxyl groups normally present in the
cellulose material. The higher the degree of regeneration, the more
stability is to be found in the resulting beads. It some cases,
wherein enzymes are to be immobilized on the cellulose bead
carriers, it is desirable to convert the hydroxy or substituting
groups into functional chemical groups, such as amino groups, which
facilitate enzyme attachment.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration of the particle size distribution of the
porous beads.
FIG. 2 is a scanning electromicrograph of a porous cellulose
bead.
FIG. 3 is a plot of the pressure-drop-characteristics of the porous
cellulose beads.
FIG. 4(A) is a scanning electron micrograph of the surface of a
porous cellulose bead (20,000x).
FIG. 4(B) is a scanning electron micrograph of the interior of a
porous cellulose bead (20,000x).
Reference is made to FIG. 1 which illustrates the size distribution
of the final porous beads obtained by distributing (by spraying) a
solution of cellulose derivative through a spray nozzle, according
to the detailed procedure outlined hereinbelow. Beads which are
either too large or too small, depending upon the intended end use,
may be collected and re-dissolved in the appropriate solvent, if
desired. Generally speaking, if employed in a column type chemical
reactor, beads of a uniform size are preferred. The desired
particle size may vary depending on the projected use of the beads,
e.g. the type of enzyme to be immobilized.
The porous cellulose beads prepared by the process described above
generally have a very high porosity and a controlled pore size
ranging from 0.05 to 30 microns. When a cellulose-to-solvent ratio
of 1:10 (weight/volume) is used in preparing the cellulose/solvent
solution, the final beads formed have a high porosity of about 90%
void. A scanning electromicrograph of a porous cellulose bead
prepared by the process is shown in FIGS. 2, 4(A) and 4(B). From
these views, one can observe several important features of the
beads produced. Firstly, it can be seen that the beads are
generally spherical in shape and porous openings are uniformly
distributed over the surface of the beads. For most applications,
this is desirable because it can provide an immobilized enzyme
catalyst of uniform activity. The void phase of the cellulose beads
is continuous. This is a desirable feature because a discontinuous,
discrete "bubble" would result in useless and nonaccessible dead
space in an immobilized enzyme system. Thirdly, there is no hard
"skin" at the bead surface. A hard skin will cause serious
diffusional hindrance. Finally, the pore sizes are quite uniform.
As a result, all of the interior surface area of the internal void
spaces of the beads will be accessible for enzyme immobilization
and for enzyme catalyzed reactions. Both the high porosity and
other noted features have made the porous cellulose beads of this
invention uniquely suited for use in immobilization of enzymes and
other biologically-active agents.
An important property of an enzyme carrier is the pressure drop it
causes at various liquid flow rates through an enzyme reactor
containing the carrier. For example, DEAE-cellulose is currently
used in industry and an enzyme carrier for the conversion of
glucose into fructose. For DEAE-cellulose, the pressure drop is
very high and consequently only shallow beds can be used to obtain
a reasonable rate of fluid flow. The pressure drop characteristics
of the porous cellulose beads of this invention in a packed column
operation is shown by Curve A in FIG. 3. The nominal linear flow
velocity is calculated by dividing the volumetric flow rate of the
feed liquid to the column by the column cross-sectional area. In
practical operations, the nominal linear flow velocity in
industrial column reactors will be less than 0.5 cm/sec. For
example, with a reactor column of two feet (60.96 cm) inside
diameter, a linear velocity of 0.5 cm/sec is equivalent to a
volumetric flow rate of 1389 gal/hr (5254 liters/hr). In a typical
industrial operation for producing fructose from glucose, the sugar
concentration in the feed is about 5 lb. sugar/gallon. The above
flow rate will yield more than 60 million pounds of the product per
2 feet column per year. Because of the residence time requirement
of the enzymatic reaction, the linear flow rate is usually less
than 0.5 cm/sec. Therefore, it can be seen that the porous
cellulose beads of this invention do not pose any serious
engineering problems with regard to pressure drop, when used in
column type chemical reactors as a carrier to which enzymes and
other biologically-active agents can be immoblized. When the porous
cellulose beads, after proper derivatization, are used for other
potential applications (e.g. removal of tannin from fruit juice,
wine or beer as well as metallic ions from dilute solutions) the
liquid flow rate through a reactor column could be much larger than
that of 0.5 cm/sec cited here.
The flow characteristics and other physical and mechanical
properties of the porous cellulose beads can be improved by
cross-linking with bi- and/or multi-functional compounds. Curve B
in FIG. 3 shows the pressure drop requirement of the porous
cellulose beads after the treatment with tolylene-2, 4-diisocyanate
and enzyme immobilization. Above a nominal linear velocity of 2
cm/sec, the untreated cellulose beads (Curve A) become compressed
and deformed considerably, resulting in a drastic increase of the
pressure drop. Curve B concaves upward only slightly indicating
little deformation, if any, of the treated beads.
Treatment of the porous cellulose beads with a cross-linking agent,
either before or after hydrolysis of the beads, results in an
increase of their physical strength. Attachment of enzymes onto the
beads will also increase their physical strength. After treatment
with, for example, a diisocyanate (e.g., tolylene-2, 4-diisocyanate
or hexamethylene diisocyanate), the beads in fact become quite
rigid and strong. Cross-linking with epichlorohydrin also improves
the physical properties of the porous cellulose beads. The
chemistry of cross-linking of polysaccharides, including cellulose
and starch, is a well-developed branch of physical science. Other
suitable cross-linking agents among others include formaldehyde in
hydrochloric acid solution or glutaraldehyde. Many other
carbohydrate cross-linking agents are well known, as shown, for
example, by Jones et al, U.S. Pat. No. 3,905,954.
In general, the porous beads of the present invention are prepared
according to the following steps:
a. a hydrolyzable form of cellulose is dissolved in an inert
organic water-miscible solvent in a controlled ratio of cellulose
derivative-to-solvent which is generally in the range of 1:20 to
1:3 (weight:volume) to produce a solvent solution. The solvent
should be wholly or substantially miscible with the precipitation
solution and the density of the solvent solution should be
sufficient that upon contact with the precipitation solution, the
solvent becomes readily miscible with the precipitation solution
and the cellulose derivative precipitates therein.
b. a solvent solution is distributed (e.g., by spraying) in the
form of droplets into a precipitation solution. Upon contact with
the precipitation solution, which may contain a surfactant, the
solvent is dispersed within the solution media and porous beads of
the cellulose material form as they coagulate and precipitate to
the bottom of the tank holding the precipitation solution. The
cellulose derivative solution may suitably be sprayed under
pressure through an atomizing nozzle into a precipitation solution
bath. If desired, the bath may be agitated to enhance the formation
of the beads.
c. the precipitated beads, after being washed, are then hydrolyzed
in order to regenerate cellulose, thereby providing a porous
cellulose bead having active sites for enzyme attachment. If
desired, in order to increase the stability of the porous beads or
provide suitable reaction sites, one can chemically modify the
beads in a number of ways. For example, the beads may be
cross-linked in order to provide greater stability and increased
physical strength. Also one can chemically substitute either
positively-charged or negatively-charged groups to alter the
surface-absorption properties of the cellulose bead. The cellulose
itself is generally hydrophilic and, thus, by altering the reaction
sites thereof, one can alter its hydrophilic properties.
The present invention further provides for a method by which
enzymes and other biological active agents may be immobilized by
attachment onto the porous cellulose beads described hereinbefore.
For example, one may convert porous cellulose beads, as described
above, to diethylaminoethyl (DEAE) cellulose by reacting said beads
with N,N-diethyl 2-chloroethylamine hydrochloride in a conventional
manner. Beads so obtained contain DEAE-cellulose and were
successfully used to attach glucose isomerase, derived from a
streptomyces culture. We have also employed a procedure involving
cyanogen bromide to immobilize the glucose isomerase.
Another procedure for enzyme immobilization on the porous cellulose
beads involves the use of tolylene-2,4-diisocyanate. Diisocyanate
was employed to cross-link cellulose to improve the physical
strength of the porous beads. However, we have found that the
porous cellulose beads of the present invention when treated with
diisocyanate, can immobilize enzymes on the surface thereof by
simply mixing the diisocyanate-treated beads together with an
enzyme solution. For example, when glucoamylase was used, the
diisocyanate beads attached more than 1000 international units of
the enzyme per gram of dry beads. While not wishing to be limited
in any way by the following theory, it appears that when dry porous
cellulose beads are in dry acetone with tolylene-2,4-diisocyanate
in the presence of a catalyst (for example, triethylamine), a
considerable degree of cross-linking occurs between cellulose
molecules in light of the improved physical strength of the beads.
After a sufficient length of time for reaction, the beads were
washed with dry acetone to remove free diisocyanate residues. The
cellulose beads appear to possess a large number of attached
isocyanate groups. Upon mixing the treated beads with an aqueous
enzyme solution, enzyme molecules appear to be covalently bonded to
the cellulose beads through the isocyanate groups. It has also been
found that washing the treated beads with water results in
converting isocyanate groups to amino groups. In such a manner, we
were successful in immobilizing an enzyme, glucoamylase, to the
amino cellulose beads with glutaraldehyde, an agent well known for
its capability of reacting and cross-linking amino groups (on the
beads and the enzyme).
The porous celulose beads produced in accordance with the present
invention also find use in the separation and purification of
enzymes, proteins, nucleic acids and the like. The porous cellulose
beads produced in accordance with the process of the present
invention may be derivatized to produce DEAE-porous cellulose beads
which possess excellent flow properties and yet are able to
efffectively separate enzymes, proteins, nucleic acids and the like
as effectively as current commercial products according to the
technique known as column chromatography.
Also, one may derivatize the porous cellulose beads of the present
invention (in situ) with groups other than DEAE. Thus, the porous
cellulose beads of the present invention are applicable for a wide
variety of specific applications. For example, one can attach a
specific functional group to the porous cellulose beads and the
then derivatized beads may be used to, for example, remove tannin
from fruit juice by passing the juice through a bed of the
derivatized porous cellulose beads with protein.
In a similar fashion, one may remove metallic ions from dilute
solutions containing same. Such a method would provide for the
recovery of valuable metallic ions (i.e., copper ions and gold
ions) from dilute mining solutions, and would find particular
applicability to current solution mining techniques whereby metals
are extracted from ores by acid solutions.
The following examples are offered to more fully describe the
invention, but are not to be construed as limiting the scope
thereof:
EXAMPLE I
Fifty grams of cellulose acetate (Visc 3 from Eastman Kodak
Chemicals) were dissolved in 400 ml of solvent A (composed of
acetone and dimethyl sulfoxide in a volume ratio of 6-to-4) to form
a 12.5% (weight/volume) solution. With a spray gun (paint sprayer
from Sears Roebuck & Co.), the cellulose solution was then
sprayed at an air pressure of 20 psi as fine droplets into a water
tank containing 40 gallons of water and four drops of common
household detergent. Upon contacting the surface of the water, the
cellulose acetate droplets coagulate into porous beads and sink to
the bottom. The porous beads were then collected and washed. The
washed beads were then deacetylated with about a 0.15 N of sodium
hydroxide overnight at room temperature. The deacetylated beads
were then washed and suction-dried, yielding a porous cellulose
bead having a void space greater than 50% by volume ready for use
in enzyme immobilization. FIG. 1 illustrates the size distribution
of the porous beads obtained. Electron micrographs revealed that
the beads were generally spherical, with the interior and surface
thereof having the same structure. The pore sizes were quite
uniform and the pores were distributed uniformly throughout the
entire bead as illustrated in FIGS. 2, 4(A) and 4(B). The pore size
of the beads was determined from scanning electron micrographs. The
scanning micrographing requires dry samples and since the drying of
the beads in air results in a size shrinkage, the beads were dried
by the critical point technique with liquid carbon dioxide. The
pore size was determined to be about 1000 A.
EXAMPLE II
Using a 10% (weight/volume) cellulose acetate solution in solvent
A, according to the process of Example I, porous beads were also
formed and were suitable for use in enzyme immobilization.
EXAMPLE III
A 10% (weight/volume) cellulose acetate (Visc 3 from Eastman Kodak
Chemicals) solution was prepared in solvent B (acetone and
formamide in a volume ration of 7-to-3). The cellulose acetate
solution was then sprayed and hydrolyzed according to the procedure
in Example I above. Highly porous cellulose beads were obtained
having a void space greater than 50% by volume.
EXAMPLE IV
The procedures outlined in Example II, above, were repeated using a
solution prepared with cellulose acetate of Visc 45 type (available
from Eastman Kodak Chemicals). Porous beads were also obtained
having excellent properties for enzyme immobilization.
EXAMPLE V
The procedures outlined in Example II, above, were carried out
using a 10% weight/volume solution of cellulose triacetate
(available from Eastman Kodak Chemicals) in solvent A. The beads
resulting therefrom exhibited excellent porosity for enzyme
immobilization. As we have noted, cellulose can be used as a
supporting material for the immobilization of enzymes and other
biologically active agents. Many workers have chosen cellulose as a
support because cellulose is inexpensive, chemically stable, and it
is resistant to microbiological contamination. Also, cellulose has
three hydroxyl groups on each anhydro-glucose unit which provides
high versatility as well as large capacity for the immobilization
of a desired substance.
The major disadvantage of using cellulose as a supporting material
is that cellulose has a fibrous shape and lacks the necessary
mechanical strength. Reactors packed with cellulose have poor flow
properties, develop severely high pressure drop, and sometimes
channelling. To overcome these problems, we prepared cellulose into
a bead form according to the present invention which exhibited a
better mechanical strength and provided enhanced flow properties
than prior materials. However, since the structure of our cellulose
beads differs from that of regular cellulose, the loading of
enzymes and stability of the immobilized enzymes may differ from
that with regular cellulose. The chemistry involved in the
preparation of immobilized enzymes not only affects the loading and
stability of the enzyme on the cellulose beads, but also affects
the mechanical strength of the cellulose beads. Any chemical
procedures for immobilization of enxymes, which increase mechanical
strength of cellulose beads, would improve the flow properties in a
reactor, as will be apparent from the examples.
EXAMPLE VI
One gram of porous cellulose beads, produced according to Example
I, was dispersed in 15 ml water which was adjusted to pH 11.5 with
sodium hydroxide and kept at a constant temperature of 20.degree.
C. One gram of cyanogen bromide was added to this dispersion. The
pH was maintained at 11.5 with 1 N NaOH. After 15 minutes, the
beads were washed with a phosphate buffer (0.1 M) at pH = 7.0 and
0.degree. C. Fifteen ml of glucoamylase solution (30 mg/ml) were
then added to the beads. The mixture was left overnight. The beads
so prepared contained 1830 units of enzyme activity per gram dry
weight of cellulose bead at 60.degree. C. using 5% maltose as
substrate. One unit of enzyme activity is defined to be that which
produces one micromole of product per minute.
EXAMPLE VII
Porous cellulose beads (0.2 gm), obtained as in Example I, were
dispersed in 5 ml acetone. 0.2 ml triethylamine was added to the
dispersion as was 0.2 ml of tolylene-2,4-diisocyanate. After 30
minutes, the beads were washed with acetone and then an acetate
buffer at pH 4.75. Five ml of glucoamylase solution (25 mg/ml) were
added. The enzyme was thereby immobilized on the beads with an
activity of 2,000 units/gm cellulose beads.
EXAMPLE VIII
Two hundred mg glucose isomerase in maleic acid buffer solution was
immobilized onto 2 gm of cellulose beads by the same procedure as
described in Example VII. The cellulose beads contained 90 units of
enzyme activity per gm of cellulose beads at 60.degree. C. using 9%
fructose as the substrate.
EXAMPLE IX
Three hundred mg of invertase in 5 ml of acetate buffer were
immobilized onto 0.5 gm of porous cellulose beads using the
procedure described in Example VII. The cellulose beads contained
3000 units activity per gm of cellulose used.
EXAMPLE X
Fifty mg of lactase in phosphate buffer (pH = 7.0) were immobilized
onto 0.5 gm cellulose beads using the procedure, described in
Example VI. The resulting cellulose beads contained about 80 units
enzyme activity per gm of cellulose beads at 30.degree. C. using 1%
lactose as substrate.
EXAMPLE XI
Five hundred mg of glucose isomerase were dissolved in 150 ml
maleic acid buffer (0.01 M, pH = 5.5). The enzyme soltuion was
pumped through 5 gm porous crosslinked cellulose beads prepared as
described in Example XVI. The DEAE cellulose beads thus contained
100 units of enzyme activity per gm of beads.
EXAMPLE XII
One-quarter gm of porous cellulose beads, produced in accordance
with Example I, was soaked in 3% of glutaraldehyde and 0.1 M
MgCl.sub.2. After drying, using vacuum suction on a Buchner funnel,
the samples were heated at 80.degree. C. for 30 minutes. Five ml of
glucoamylase (25 mg/ml) were added to the beads. After standing
overnight, the beads thus prepared contained about 200 units of
enzyme activity per gm of dry cellulose beads.
EXAMPLE XIII
One gm of porous cellulose beads was cyanoethylated with 10 ml
acrylonitrile (C = CC.tbd.N) at 50.degree. C. The so-treated
cellulose beads were then treated with hydorxylamine at a pH 6.5 -
6.7 at 50.degree. - 100.degree. C. for 4 hours. The resulting
modified porous bead product contained ##STR1## groups and is
suitable for absorbing heavy ions such as ferric, ferrous, and
cupric.
EXAMPLE XIV
A suspension of 2.5 gm porous cellulose beads was treated with 2.5
ml hexamethylene diisocyanate and triethylamine, followed by
hydrolysis in water. The product was then treated with 50 ml of 0.5
M 0-methyl iso-urea at pH 5. The product obtained has the following
funtional group: ##STR2## which is useful as an anionic ion
exchanger.
EXAMPLE XV
Five grams of porous cellulose beads, obtained according to Example
I, were added to 100 ml of 36% formaldehyde and 200 ml of 37%
hydrochloric acid. After standing for 11/2 hours at room
temperature, the beads were filtered and subsequently washed with
water and 0.2% sodium carbonate solution. The beads were then dried
at 75 to 80.degree. C. The resulting cross-linked porous cellulose
beads exhibited strong physical strength.
EXAMPLE XVI
Three grams of porous cellulose beads were cross-linked by
formaldehyde according to the process in Example XV. The beads were
then treated with 3 grams of 2-chlorotriethylamine. After heating
the mixture for a period of 35 minutes at a temperature of 80 to
85.degree. C., the beads were then washed sequentially with sodium
chloride, sodium hydroxide, hydrochloric acid, water and ethanol.
The cross-linked porous DEAE cellulose beads so obtained exhibited
excellent porosity having a void space greater than 50% by
volume.
EXAMPLE XVII
A dispersion was formed of 0.5 grams porous cellulose beads in 5 ml
of 0.2 N sodium hydroxide and 5 ml epichlorohydrin. The dispersion
was then heated for several minutes to a temperature of 80.degree.
C. Subsequently, the beads were washed and the cross-linked porous
beads so treated exhibited greater strength than the porous
cellulose beads prior to crosslinking. Wet cellulose beads,
obtained according to the procedure of Example I, were washed in
acetone. The washed beads were then suspended in dry acetone
containing 0.6 ml of triethylamine for each gram of cellulose.
Tolylene-2,4-diisocyanate, in an amount of 1.6 ml per gram of
cellulose beads was added to the suspension at 0.degree. C. After a
period of 30 minutes, the beads were washed with dry acetone and
subsequently filtered. The resulting porous cellulose beads contain
isocyanate-reactive groups which could then be hydrolyzed to an
amino group by the addition of water.
EXAMPLE XVIII
Two tenths g of the cellulose beads produced as in Example I were
suspended in 10 ml of distilled water, the pH was adjusted to 11.5
by the addition of 1 N NaOH at 20.degree. C. Two tenths g of CNBr
was added to the cellulose beads suspension, a small portion at a
time, and the pH was maintained by an auto-titratometer with 1 N
NaOH. After 20 minutes the beads were washed with ice cold
distilled water and an appropriate buffer solution. Enzymes
dissolved in a proper buffer solution were added to the washed
cellulose beads. Cellulose (Solka floc) used in this method was
mercerized with 18% (w/v) NaOH for 4 hours then washed with
distilled water.
EXAMPLE XIX
Two g of suction dried cellulose beads of Example I were washed
with acetone to remove moisture and were suspended in 10 ml of
acetone. One tenth ml of triethylamine or dibutyltin diacetate were
added as catalyst. One tenth ml of tolylene-2,4-diisocyanate or
hexamethylene diisocyanate were added to the cellulose bead
suspension. After 45 minutes of reaction at ambient temperature,
the cellulose beads were washed with acetone to remove excess
diisocyanate and water was then used to wash the cellulose beads to
remove acetone. Enzymes dissolved in an appropriate buffer solution
were added to the cellulose beads. The cellulose beads were stored
at 4.degree. C overnight.
EXAMPLE XX
Aryl diisocyanate was attached to cellulose beads as described in
Example XIX. Before the enzyme solution was added, the cellulose
beads were suspended in distilled water. One tenth ml of
triethylamine was added to catalyze the reaction between isocyanate
and water to form aryl amine cellulose beads. The arylamine
derivative was then diazotized with NaNO.sub.2 in HCl. Enzymes
suspended in a proper buffer solution were then attached to the
cellulose beads.
EXAMPLE XXI
Diisocyanate attached on the cellulose according to Example XIX
reacts with water to form amino group with or without a tertiary
amine as catalyst. Glutaraldehyde is used to couple the enzyme onto
the cellulose beads by crosslinking amino groups on enzymes and on
cellulose beads.
EXAMPLE XXII
Two g of suction-dried beads produced in accordance with Example I
were suspended in 10 ml of 3% glutaraldehyde which was 0.1 M in Mg
Cl.sub.2. The suspension was heated at 100.degree. C for 30
minutes. The cellulose beads were then washed with distilled water.
Enzymes dissolved in an appropriate buffer solution were added to
the cellulose beads. The reaction was allowed to continue overnight
at 4.degree. C.
EXAMPLE XXIII
One g of cellulose beads produced according to the procedure of
Example I was refluxed with 10 ml of 10%
3-aminopropyltriethoxysilane in toluene for 4 hours. The cellulose
beads were then filtered and washed with acetone. 2.5% (w/v)
glutaraldehyde solution in 0.1 M phosphate buffer pH = 7.0) was
added to the cellulose beads at ambient temperature for one hour
with occasional stirring. The cellulose beads were then washed
thoroughly with water and an appropriate buffer solution. Enzymes
dissolved in an appropriate buffer solution were added to the
cellulose beads. The reaction was allowed to continue overnight at
4.degree. C.
EXAMPLE XXIV
Porous cellulose beads produced in accordance with the procedure of
Example I were first cross-linked by 36% formaldehyde and 37% HCl
with a volume ratio of 5 to 1. The crosslinked cellulose beads (5 g
dry weight) were suspended in 50 ml of cold 1.5 N NaOH solution.
Six g of 2-Chlorotriethylamine hydrochloride were added to the
cellulose beads. The mixture was then heated at 80.degree. -
85.degree. C for 35 minutes. The mixture was cooled in an ice bath
and filtered. The cellulose beads were washed with 500 ml of 2M
NaCl and then were washed with 200 ml of 1N NaOH and 200 ml of 1N
NaOH, alternately for three times. After washing with another 200
ml of 1N NaOH, the cellulose beads were washed with distilled water
until the pH of washing water became neutral. The reaction with
2-chlorotriethylamine hydrochloride was repeated again for a higher
degree of substitution. Enzymes dissolved in an appropriate buffer
were added to the cellulose beads for overnight at 4.degree. C.
EXAMPLE XXV
Hexamethylene diisocyanate was attached to cellulose beads as
described in Example XIV and then the isocyanate groups were
hydrolized to form amino group as described in Example XX. O-methyl
isourea was added to the cellulose beads to incorporate the
guanidino function into the derivatized beads.
Examples XVIII through XXIII provide for immobilization of enzymes
by covalent bonding, whereas Examples XXIV and XXV utilize ionic
adsorption. Glucoamylase, glucose isomerase and invertase were
loaded onto various of the beads of Examples XVIII to XXV and the
amount of enzyme loading measured.
The enzymes immobilized by covalent bonding were washed with 2M
NaCl solution to remove the absorbed enzymes. Some properties of
the immobilized enzymes are shown in Table 2. It indicates that the
same chemistry used for regular cellulose can also be used for
cellulose beads. The fact that cellulose beads have higher enzyme
loading capacity than regular cellulose may indicate a larger
surface area in the porous cellulose beads.
EXAMPLE XXVI
Protein and enzymes may be separated and purified according to the
following procedure. 2 gm of glucose isomerase (Strep. albus
obtained from Miles Laboratory) was suspended in 20 ml of 0.01M
phosphate buffer (pH = 7.0) and the suspension centrifuged. The
supernatant was added to a column of DEAE-porous cellulose beads
produced according to Example XVI. The bed volume was 30 ml and the
column diameter 1.5 cm. The colume was washed with 0.01M phosphate
buffer (pH = 7.0). The column was eluded with NaCl gradient
solution in 0.01 M phosphate buffer. Glucose isomerase began to
elude out of the column in the NaCl fractions with concentrations
ranging from 0.25 to 0.45 M.
Table 2 ______________________________________ Enzyme Loading on
Porous Cellulose Beads Enzyme loading on cellulose, IU*/g
(calculated from initial Method of reaction rate) Immobili- Porous
zation regular cellulose Assay Enzymes Example cellulose beads
conditions ______________________________________ XVIII 820 1,8000
10% Maltose, 60.degree. C XIX 550 " Glucoamylase XX 275 10%
Maltose, (A. Oryzae) 40.degree. C XXI 530 5% Maltose, 60.degree. C
XXII 80 190 10% Maltose, 60.degree. C XXIII 200 " Glucoamylase XXIV
3,000 9,000 " (A. Niger) XXV 1,000 " XIX 90 0.5M Fruc- Glucose
tose, 60.degree. C Isomerase xXIV 300 " (Strep. albus) XXV 160 "
Invertase XIX 1,140 0.125M (Candida Sucrose,45.degree. C utilis)
XXIV 2,000 " XXV 1,840 " ______________________________________ *IU
- international units
EXAMPLE XXVII
The porous cellulose beads of Example XIII are added to a 0.05 M
sodium acetate solution (pH = 5.2) which contains 1,600 ppm of
cupric ion. After one hour, the cellulose beads picked up 6.3%
cupric ion by weight of the beads.
We have also found that when the porous cellulose beads of the
present invention are dried and/or heated, e.g. at 100.degree. C
prior to use, the resulting beads exhibit an increased physical
strength.
The invention, in its broadest aspects, is not limited to the
specific details shown and described, but departures may be made
from such details within the scope of the accompanying claims
without departing from the principles of the invention.
Furthermore, the invention may comprise, consist, or consist
essentially, of the hereinbefore-recited materials and steps.
* * * * *